Occurrence of resorcyclic acid lactones in porcine urine: discrimination between illegal use and contamination

Figures & data

Figure 1. Structures and acronyms of resorcyclic acid lactones and probable pathways of phase I metabolism (Kennedy et al. 1998).

Figure 2. UHPLC–ESI–MS/MS chromatogram corresponding to a standard mixture solution (1 µg L⁻¹) of targeted compounds obtained under optimal gradient elution conditions. (β-ZAL), β-zearalanol m/z 321.3; (β-ZOL), β-zearalenol m/z 319.3; (α-ZAL), α-zearalanol m/z 321.3; (α-ZOL) α-zearalenol m/z 319.3; (ZAN) zearalenone m/z 319.3; (ZEN) zearalenone m/z 317.3.

Table 1. Performance characteristics of the developed LC − MS/MS per analyte in porcine urine (n = 7).

Parameter		β-ZAL	β-ZOL	α-ZAL	α-ZOL	ZAN	ZEN
Recoveries (%)		60	57	62	60	60	57
Linearity (R^2)	(0.5–5 µg L^−1)	0.999	0.999	0.999	0.989	0.999	0.994
Concentration (µg L^−1)	Low level	0.5	0.5	0.5	0.5	0.5	0.5
	Medium level	1	1	1	1	1	1
	High level	1.5	1.5	1.5	1.5	1.5	1.5
Trueness (%)	Low level	103	102	99	68	99	88
	Medium level	92	107	98	79	107	109
	High level	96	103	99	93	104	111
Repeatability (RSD,%)	Low level	7	10	7	30	17	38
	Medium level	14	6	5	27	16	15
	High level	13	7	7	14	25	13
Confirmation rate	Low level	7/7	7/7	7/7	7/7	7/7	7/7
	Medium level	7/7	7/7	7/7	7/7	7/7	7/7
	High level	7/7	7/7	7/7	7/7	7/7	7/7
CCα		0.62	0.68	0.62	0.85	0.80	1.09

Table 2. Feed contaminated by zearalanone (Animal study A): concentrations found in urine (µg L⁻¹).

	Treated animal						Non treated animal
	Fusarium tox.			Forbidden subs.			Fusarium tox.			Forbidden subs.
Day	α-ZOL	β-ZOL	ZEN	α-ZAL	β-ZAL	ZAN	α-ZOL	β-ZOL	ZEN	α-ZAL	β-ZAL	ZAN
1	−	−	−	−	−	−	−	−	−	−	−	−
2	−	−	−	−	−	−	−	−	−	−	−	−
3	−	−	6.8	−	−	−	0.51	−	8.6	−	−	−
4	0.54	−	8.8	−	−	−	0.38	−	10	−	−	−
5	0.72	−	4.7	−	−	−	0.51	−	5.9	−	−	−
8	8.6	0.47	66	39	3.02	36	1.05	−	9.6	0.56	−	0.49
9	6.3	−	18	12	1.86	14	1.1	−	8.7	0.49	−	1.2
10	4.1	0.33	5.5	3.6	1.00	5.9	4.2	0.75	14	−	0.09	0.45
11	14	1.1	28	14	3.09	20	4.1	0.71	15	0.21	0.61	1.8
12	18	1.3	23	14	3.1	18	9.6	1.2	16	−	0.25	0.78
15	19	1.1	27	12	3.9	17	2.9	0.57	13	−	0.02	0.21
16	6.4	0.58	17	16	1.8	15	2.9	0.57	8.9	−	0.00	0.07
17	16	0.91	21	14	2.7	22	4.2	0.7	22	−	0.12	0.43
18	15	1.0	25	10	4.2	13	6.3	1.2	25	0.38	0.58	0.64
19	18	1.2	22	13	5.6	17	3.6	1.0	22	0.28	0.37	0.45
22	12	3.9	20	12	3.9	18	−	−	−	−	−	−
23	11	2.6	20	11	2.6	16	20	2.3	20	−	0.04	0.17
24	25	15	49	25	15	54	−	−	−	−	−	−
25	18	5.7	35	18	5.7	33	4.8	0.67	23	−	0.02	0.11
26	21	6.4	39	21	6.4	36	−	−	−	−	−	−

Table 3. α-Zearalanol administration (Animal study B): concentrations found in urine (µg L⁻¹).

	Treated animal						Non treated animal
	Fusarium tox.			Forbidden subs.			Fusarium tox.			Forbidden subs.
Day	α-ZOL	β-ZOL	ZEN	α-ZAL	β-ZAL	ZAN	α-ZOL	β-ZOL	ZEN	α-ZAL	β-ZAL	ZAN
1	−	−	−	−	−	−	−	−	−	−	−	−
2	−	−	−	−	−	−	0.26	−	0.95	−	−	−
3	−	−	4.9	−	−	−	−	−	5.8	−	−	−
4	−	−	−	−	−	−	1.04	−	9.7	−	−	−
5	−	−	3.5	−	−	−	−	−	−	−	−	−
8	0.82	−	20	255.0	50	154	2.2	−	10	2.0	0.04	3.6
9	0.35	−	11	116.0	75	81	−	−	−	0.80	−	0.43
10	2.1	0.36	7.3	33.0	19	86	2.4	0.21	1.1	0.09	0.05	0.04
11	2.5	0.43	5.0	9.5	6.3	30	3.3	0.34	3.9	−	0.06	0.11
12	3.7	0.56	6.4	4.7	2.9	16	3.6	0.37	4.2	−	0.06	0.24
15	1.5	0.19	6.2	759	84	474	3.3	0.26	5.8	0.04	0.10	0.67
16	1.9	0.21	3.5	69	37	197	−	−	−	−	−	−
17	1.5	0.17	3.5	17	9.6	46	1.9	0.10	1.5	−	0.00	−
18	2.2	0.61	7.2	14	7.3	23	3.1	0.58	6.2	0.39	0.52	0.64
19	2.5	0.54	4.6	5.6	3.4	9.4	2.9	0.47	4.9	0.28	0.37	0.47
22	1.4	0.26	6.9	655	39	488	1.5	0.27	6.3	−	0.00	0.17
23	0.41	−	1.5	44	12	69	2.4	0.24	4.8	−	0.03	0.02
24	1.8	0.39	12	202	56	331	4.3	0.26	13	0.57	0.36	3.8
25	2.3	0.44	8.7	98	30	176	1.9	0.12	2.6	−	0.01	−
26	2.2	0.35	10	63	16	133	2.5	0.25	6.7	−	0.04	0.38

Figure 3. Profile of the six RALs ((β-ZAL), β-zearalanol; (β-ZOL), β-zearalenol; (α-ZAL), α-zearalanol; (α-ZOL) α-zearalenol; (ZAN) zearalenone; (ZEN) zearalenone) determined by UHPLC–MS/MS analysis of the collected urine samples at feed contaminated by zearalenone (Study A). Samples are labelled according to the collection day in the study.

Figure 4. Profile of the six RALs ((β-ZAL), β-zearalanol; (β-ZOL), β-zearalenol; (α-ZAL), α-zearalanol; (α-ZOL) α-zearalenol; (ZAN) zearalenone; (ZEN) zearalenone) determined by UHPLC–MS/MS analysis of the collected urine samples at α-zearalanol administration (Study B). Samples are labelled according to the collection day in the study.

Figure 5. Obtained ratio based on the metabolite pattern of α/β-ZAL + ZAN vs α/β-ZOL + ZEN for (A) Animal study A: Feed contaminated by zearalenone and (B) Animal study B: α-Zearalanol administration. Blue dash lines correspond to the values of control animals while continuous lines to treated animals.

Kennedy DG, Hewitt SA, McEvoy JDG, Currie JW, Cannavan A, Blanchflower WJ, Elliot CT. 1998. Zeranol is formed from Fusarium spp. toxins in cattle in vivo. Food Additives & Contaminants. 15(4):393–400. doi: 10.1080/02652039809374658.

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